S121: CD123-CD33 COMPOUND CAR-T CELLS WITH NOVEL ANTIGEN BINDING DOMAINS PROVIDE A NEW HOPE FOR THE TREATMENT OF ACUTE MYELOID LEUKEMIA
Authors: Z. Wang, Y. LU, S. Qiu, M. Wang, D. Xiong, J. Wang
Journal: HemaSphere (2022)

Background: CAR-T therapy needs to be optimized to treat myeloid malignancies. Antigen-escape mediated relapse after CAR-T treatment is the vital mechanism responsible for the nondurable response. Targeting CD33 and CD123 simultaneously could cover almost all patients with AML. The off-target cytotoxicity of CD123 CAR-T and CD33 CAR-T, especially on HSPCs, has always been concerned. Several researchers have explored this issue and the conclusions are inconsistent in different studies. Aims: To improve the outcome of relapse and/or refractory acute myeloid leukemia and explore the influence of CD123-CD33 CAR-T on hematopoiesis. Methods: We develpoed a panel of anti-CD123 monoclonal antibodies (mAbs) using hybridoma technology, named 6E11, 8D7, 12H7 and 13C3 and incorporated each of the four scFvs into 4-1BB/CD3ζ signaling domain to generate 4 second generation CARs. T cells were infected with lentiviral supernatants to generate CAR-T cells. Comparative analysis were made from the expression property of CAR protein, in vitro and in vivo anti-leukemia efficacy to screen out the best candidate CD123 CAR. Next, we generated a CD123-CD33 dual targeting CAR in a compound way. We established an escape-simulation model to explore whether the compound CAR-T could reduce the risk of relapse associated with antigen loss. Flowcytometry analysis, CFU test and single cell RNA sequecing methods were jointly used to explore the influence of our CD123-CD33 CAR-T on hematopoiesis. Results: CD123 CAR constructs with four different novel scFvs differed in CAR protein expression ratio. A changing trend of CAR proteins from dropping to rising after encountering target cells was observed in 3 CAR-T cell group except in 8D7 CAR-T. 13C3 CAR was the most suitable construct from the perspective of basic and dynamic expression. 123CAR-T cells with different scFvs mediated variable anti-leukemia effect in vitro and in vitro. 123CAR and 33CAR proteins expressed efficiently on the compound CAR-T cell surface without codon optimization. The compound CAR-T showed superiority in the treatment of antigen-escape mouse model. FCM analysis showed no significant difference of residual CD34+ cells proportion between NCT cell group and compound CAR-T group. scRNA-seq analysis showed that the most undifferentiated HSC population barely changed among each group. GSEA revealed that the HSC population in CAR-T groups had no difference in the enrichment of quiescence, cell cycle, G2M checkpoint related genes compared with that of NCT group. In addition, no downregulation/enrichment of TGF-beta signaling pathway/ PI3K-AKT-mTOR signaling pathway was observed in compound CAR-T group compared with NCT group.The CFU assay demonstrated that there were still quite numbers of hematopoietic colonies formed in compound CAR-T treatment groups. Image:Summary/Conclusion: Taken together, in this study we developed a panel of second generation CD123 CARs with novel scFvs on the basis of 4 novel anti-CD123 mAbs. We proved that scFv sequence has a significantly impact on CAR expression both originally and dynamically and further affects CAR-T function, addressing the importance of scFv development in CAR-T therapy refining. The compound CAR-T in our study minimize the risk of antigen-loss mediated escape. Notably, wildtype DNA sequence of cCAR without codon optimization brings no potential risk such as immunogenic complications. scRNA-seq and CFU assay convincingly demonstrated that the CD123-CD33 compound CAR-T had limited toxicity on hematopoiesis. Thus, the CD123-CD33 compound CAR-T with novel scFvs provides new hope for R/R AML patients.